| ObjectiveIn the experimental study,it was to explore cartilage injury of knee osteoarthritis molecular mechanism of Bushen Tongluo formulae by regulating Wnt/β-catenin pathway through establishing animal model of knee osteoarthritis and effects on behavior and cartilage morphology.The clinical study was conducted to analyze efficacy and safety of Bushen Tongluo formulae in treatment of KOA patients with syndrome of liver and kidney deficiency and blood stasis effects on Wnt/β-catenin signaling pathway.Experimental researchMethod:10 male pathogen-free,healthy New Zealand white rabbits,according to random number table divided into model group 1(that was,right knee plaster fixed braking)and model group 2(that was,bilateral knee joint cavity injection of papain),5 rabbits each group.Before and after building,observe animals’ general states and record width and passive range of knee joint,Lequesne MG index assessment was used to analyze their behavior index score.The protein content of PGE-2、IL-1β、TNF-α and COMP in serum and joint fluid was determined by ELISA.42 pathogen-free,healthy New Zealand rabbits with age of 10 weeks,half male and female,according to random number table to select 6 as normal group animals first,not do any intervention.The remaining 36 experimental animals were injected papain solution by double knee joint cavity copying model of KOA.After modeling according to random number table again rabbits were randomly divided into 6 groups,namely model,western medicine,proprietary Chinese medicine,low,medium and high dose groups of traditional Chinese medicine,each group of 6 respectively.Normal and model groups were given distilled water to fill stomach,the rest to medicine group respectively for meloxicam,prevent bone hyperplasia capsule,Bushen Tongluo formulae of low,medium and high dose suspension liquid.The drug was administered one time a day for four weeks.The qualities,range of knee joint passive activity and Lequesne MG index of each group were recorded weekly.Musculoskeletal ultrasound equipment was used to evaluate the inflammatory condition of synovial membrane in experimental animals every 2 weeks.Drug intervention after 4 weeks,respectively with naked eye evaluate knee joint cartilage at tibia platform,by HE staining under light microscopy observation of knee joint cartilage morphology and by Mankin’s method to score them.Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of Wnt7α,β-catenin,Axin,GSK-3β and TGF-β1,BMP-4,BMP-7 detected by Western blot.The protein contents of P-catenin,TGF-β1,BMP-4,BMP-7 were detected by ELISA.The spleen and thymus quality of experimental animals were obtained,and their organ index was recorded and analyzed.Results:The states of all animals decreased after molding,and increased after 2 weeks.The score of Lequesne MG was normal before molding,and module 2 was slightly higher than module 1,but there was no statistically significant difference between two groups(P>0.05).The content of serum PGE-2、IL-1β、TNF-α was higher than that of module 1,and the content of COMP was higher than that of module 2,and the difference between two groups was statistically significant(P<0.05).Content of PGE-2、IL-1β、TNF-α in joint fluid of module 2 was higher than module 1,COMP content was still higher than module 2,the content of IL-1β and TNF-a between two groups was statistically significant(P<0.05),while the content of PGE-2 and COMP no statistically significant difference(P>0.05).About different groups of experimental animals,there was no statistically significant difference between the groups in baseline period(P>0.05).After molding,it decreased significantly and increased after 2 weeks.Compared with normal group,the passive activity score decreased and Lequesne MG index score increased(P<0.05).After 4 weeks of drug intervention,compared with model group,the passive activity score increased and Lequesne MG index score decreased(P<0.05).Ultrasound images showed that in normal group and baseline period of each group,the score of synovial inflammation was 0,and the score of synovial inflammation in each group was increased,which was significantly different from that in normal group(P<0.05).The inflammatory scores of each group decreased,and the differences with model group were statistically significant(P<0.05).Knee joint cartilage gross score and Mankin’s method of cartilage scores showed that compared with normal group,after building each score increased(P<0.05),compared with model group score decreased(P<0.05),high dose group decreased significantly in traditional Chinese medicine.Real time-PCR detection results:compared with normal group,the rest groups of Wnt7a and β-catenin mRNA expression level increased(P<0.05),the rest group of Axin and GSK-3β mRNA expression level decreased(P<0.05).Compared with model group,the Wnt7α,β-catenin mRNA expression levels were reduced(P<0.05),and the western medicine group was obviously.In each dosage group,Axin and GSK-3β mRNA expression level increased(P<0.05),high dose group significantly increased in traditional Chinese medicine.Western blot test results:compared with normal group,the rest groups Wnt7a andβ-catenin protein content were increased(P<0.05),TGF-β1,BMP-4 and BMP-7 protein content were reduced(P<0.05),in addition to the medium dose group of Axin and high dose group of GSK-3β levels higher than normal group,the others all decreased(P<0.05).Compared with model group,the content of Wnt7a and β-catenin in each group decreased(P<0.05),and the protein content of Axin,GSK-3β,TGF-β1,BMP-4 and BMP-7 all increased(P<0.05).ELISA test results:compared with normal group,levels of TGF-β1 protein in the other groups were increased(P<0.05),and the protein content of BMP-4 and BMP-7 decreased(P<0.05).Compared with model group,content of β-catenin in each drug group decreased(P<0.05),and the protein content of TGF-β1,BMP-4 and BMP-7 increased(P<0.05).Viscera index results:the indexes of spleen and thymus organs in each group were the largest in low dose group,but there was no statistically significant difference between the groups(P>0.05).Clinical researchMethods:60 cases of KOA patients with liver and kidney deficiency and blood stasis syndrome were collected.The patients were divided into treatment and control group by random number table,with 30 cases in each group.In control group,the treatment was given to external application of waxy medicine 1 time daily,combined with oral glucosamine sulfate 3 times,2 tablets each time.The treatment group was given a daily dose of oral Bushen Tongluo formulae therapy on basis of control group.Both groups were treated for 4 weeks.The patient’s names,gender,age,course of disease,height,weight,BMI and drug allergy were recorded in detail before treatment.Need to record before and after treatment in patients with KOA syndromes quantitative assessment,WOMAC table,knee pain VAS score,SF-36 and change of clinical routine laboratory indexes,by musculoskeletal ultrasound examination of knee joint,testing patients peripheral blood serum of Wnt7α、βP-catenin、Axin、GSK-3β、TGF-β1、BMP-4、BMP-7 protein content using ELISA were recorded and analyzed.Results:(1)General information:18 cases of female treatment group,12 male patients,age(62.30±8.94),disease duration(5.48 ±2.89),BMI(25.44±1.55)kg/m2.In control group,there were 17 female patients,13 male patients,age(59.73±9.89),and the course of disease(4.93 ±3.08),BMI(25.40 ±0.94)kg/m2.There was no statistically significant difference between groups(P>0.05).(2)Efficacy score of syndrome:the score of treatment group after treatment was less than that of control group,and difference between two groups was statistically significant(P<0.01).Before and after treatment,difference was statistically significant(P<0.01).(3)Pain VAS self and other assessment;WOMAC score and SF-36 score scale:the changes in the scores after treatment were significantly lower than those before treatment,and the difference was statistically significant(P<0.05).After treatment,treatment group was lower than control group in pain VAS assessment and total score of WOMAC,and SF-36 score increased,and difference between the groups was statistically significant(P<0.05).(4)Ultrasonic situation:after treatment,two groups of patients with knee joint synovial thickness decreases,and within the same group before and after treatment comparison difference was statistically significant(P<0.05),no obvious difference between groups compare,no statistical significance(P>0.05).(5)ELISA test results:after treatment,treatment group Wnt7a and β-catenin protein content decreased,TGF-β1,BMP-4 and BMP-7 levels increase.(6)Routine clinical laboratory examination:there were no obvious changes in the safety indicators such as blood,urine routine and hepatic and renal function,and the changes in ESR and CRP were not obvious.No adverse events occurred during drug therapy.Conclusion1.This study adopts knee joint cavity injection of papain to copy KOA New Zealand rabbits,can make them joint pain,swelling,limited activity,and PGE-2,IL-1β and TNF-a in serum and joint fluid,with advantages such as short time,low cost,good stability.2.Bushen Tongluo formulae intervention on behavior,imaging and morphological observation,can effectively increase knee joint activities,relieve pain,swelling,symptoms,reducing level of synovial inflammatory thickening articular cartilage injury situation and be improved to a certain extent,among them with medium and high dose groups curative effect more pronounced.3.Cartilage and joint fluid related protein test results suggested that through inhibition of Wnt/β-catenin signaling pathway,stimulates production of BMP/TGF-β1,reduce the content of Wnt7α,β-catenin,increase GSK-3β,Axin,TGF-β1,BMP-4,BMP-7,thus improved anabolism of KOA cartilage cells,reduced KOA cartilage damage degree,among them with medium and high dose groups curative effect more pronounced.4.By applying Bushen Tongluo formulae with treatment of liver and kidney deficiency,blood stasis resistance KOA patients,with analysis of TCM syndrome score scale and SF-36 scale,and WOMAC,VAS score and knee ultrasonic imaging findings,can improve the score of related situation,quality of life,decrease knee joint inflammatory symptoms.Through detection related proteins in patients with serum,it decreased Wnt7α,higher BMP-4,to some extent from molecular level improving its condition.This party was safe,effective and without obvious adverse reactions. |
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